The expression of proto-oncogene c-src, the cellular counterpart of the transforming gene v-src of Rous sarcoma virus, was found to be tissue specific and developmentally regulated. Instead of the commonly observed 4 kb mRNA, a smaller class of 2.8 to 3.3 kb c-src mRNAs is present in chicken skeletal muscle. Induction of the expression of the smaller c-src mRNAS concurs with the disappearance of the 4 kb RNA in muscle and occurs shortly before hatching. The smaller c-src mRNAs lack most of the tyrosine kinase domain, and most likely code for a protein other than tyrosine kinase. The 4 kb and the smaller c-src mRNAs are apparently generated by alternative splicing. The goal of this proposal is to explore this system to study the function and molecular mechanism of regulation of c-src proto-oncogene. cDNA clones of the 4 kb and 2.8 to 3.3 kb RNAs will be isolated and sequenced to reveal their sequence content and the potential product of the smaller c-src mRNAs. The exon structure and promoter sites of c-src DNA will be analyzed to elucidate the pattern of alternative splicing for those mRNAs. Expression of c- src in mouse and rat will be examined to see if similar tissue specificity and developmental regulation exists in mammalian species. Polyclonal and monoclonal antibodies against the muscle-specific c-src protein will be prepared. These antibodies will be used to identify and then to localize the c-src protein in muscle tissue. Its function will be explored by blocking its expression with anti- sense retrovirus-containing sequences unique to the smaller c-src mRNAs. Factors controlling c-src expression will be investigated at cellular and molecular levels. Effect of synaptic muscle activity on expression of the specific c-src RNAs and proteins(s) will be approached by denervation and by using neurotoxins. Potential factors interacting with promoter sequences will be analyzed by DNA gel retardation and footprinting analyses. DNAse hypersensitive and hypomethylation sites will be mapped and correlated to c-src gene activity. Potential trans-acting factors affecting RNA splicing and poly(A) site selection will be investigated by in vitro RNA processing system.